The Experiment Study For The Mechanism Of Endothelial Dysfunction By Homocysteine

Hyperhomocysteinemia is a independent risk factor for ateriosclerosis, and up to 40% of patients with coronary or cerberovascular atherosclerosis have hyperhomocysteinemia. The mechanism of endothelial dysfunction by Hey is not clear, even some experimental results are contradictory for each other. So a cause-and-effect relationship remains to be fully established.Objectives: (i).Through observation of the changes of NO , ET and activity of GSH-PX s SOD in plasma of HHcy rabbits to study the effects of oxidant stress in endothelial dysfunction; (2). TO observe whether the antioxidant ascorbic acid (VitC) could prevent hyperhomocysteinemia-induced endothelial dysfunction in rabbits and study the mechanism; (3).Through transmission electron microscope to observe the ultrastructural changes of endothelial cells, smooth muscle cells in external iliac atrteries of hyperhomocysteinemia rabbits; (4).To study the changes of NO, ET in cultured HUVEC with Hey in different concentration; ㏕hrough RT-PCR to study whether eNOS mRNAwas influenced by Hey in different concentration in cultured HUVEC.Materials and methods: (1). Experiment one: Eighteen rabbits were randomized into two groups (n=9 per group). Control group (N group) were poured water into stomach, Methionine group (M group) were poured L-methionine (5.0g/kg weight) into stomach. NO , ET and activity of GSH-PX, SOD in plasma were examined at 0, 4, 8, 12, 24 hour, at same time, EDD, EID were examined by high resolution ultrasound and external iliac arteries were observed by transmission electron microscope after 24 hours; (2). Experiment two: Rabbits were randomized into three groups (n=9 per group), normal control group (N group) received normal diet, methioine group (M group) received methioine-rich diet consisted of L-methioine 0.8g/kg weight.day, rnethioine+VitC group (MC group) received diet consisted of L-methioine 0.8g/kg weight.day and VitC 1.0g/kg weight.day. Then NO, ET and activity of GSH-PX, SOD in plasma were examined at 20, 40, 60, 80 day. At same time, EDD, EID were examined by high resolution ultrasound and external iliac arteries were observed by transmission electron microscope after 80 days. (3).Experiment three: Human umbilic vein endothelial cells were collected and cultured to the third generation. Then HUVECs were cultured with Hey in different concentration (physiological concentration 0.01mmol/L pathophysiological concentration 0.5mmol/L , pharmacological noncytotoxic concentration 2.0mmol/l_ , pharmacological cytotoxic concentration 5.0mmol/L) for 24 hours. NO, ET in culture medium were examined in 4, 6, 8, 24 hour. After 24 hours, HUVECs were collected to isolated of total RNA. With eNOS special primer, one microgram oftotal RNA was reverse transcribed into cDNA, then cDNA was amplified under standard conditions. At last, the PCR products were analysed by gel electrophoresis.Resluts: (1).In acute hyperhomocysteinemia, EDO of external iliac artery in M group were time-dependent reduced significantly than control group (0 hour, 12.53?.84%; 4 hour, 15.84?.19%; 8 hour, 1.36?.81%; 12 hour, -1.56?.13%; 24 hour, 3.57?.15%, p< 0.01 ),but EID were not affected; (2). In acute hyperhomocysteinemia, NO in plasma of M group rabbits was reduced significantly than control group (N group: 248.52 ?7.56 v- mol/L vs M group: 0 hour, 216.13?1.28 u mol/L; 4 hour, 105.93?6.57 u mol/L; 8 hour, 104.38110.03 n mol/L; 12 hour, 117.63139.64 u mol/L: 24 hour, 83.02?8.89 u mol/L, p < 0.05),but ET in plasma of M group rabbits was increased significantly (N group: 168.47?7.52pg/ml vs M group: Ohour, 187.53?7.51 pg/ml: 4 hour, 262.13197.91 pg/ml; 8 hour, 387.33135.25 pg/ml: 12 hour, 452.55l32.84 pg/ml: 24 hour, 370.26?3.72 pg/ml, p < 0.01) . There was no difference in GSH-PX, SOD between M group and C group; (3) .In chronic hperhomocysteinemia, EDO of external iliac arteries in MC group (80 day, 3.4912.07%) and M group (80 day, -9.2612.73%) wer...